I propose to combine my career interests in medical genetics and gene therapy by studying Adeno Associated Virus (AAV) vector transduction. The University of Washington is a unique place to study both genetics and gene therapy and perhaps unparalleled in its opportunities for researchers with this combined interest. AAV vectors are currently being used in 9 human gene therapy trials. Several questions remain about the fate of vector genomes after cellular entry, including the effect on the host cell chromosome and mechanisms of integration. We have shown that AAV vectors can integrate by separate pathways distinguished by the presence or absence of vector homology to chromosomal sequences. We analyzed non-homologous vector integration junctions and found sequence alterations similar to the findings of others investigating the non-homologous end joining pathway of mammalian double strand break repair (DSBR) implying that these breaks may be a substrate for vector integration. The observation that genotoxic agents that generate double strand breaks increase AAV vector transduction frequencies also supports this hypothesis. We will evaluate the role of double strand breaks in AAV vector transduction by generating breaks in human cells using conditional induction of the I-Scel endonuclease, and evaluate transduction frequencies in the presence and absence of chromosomal breaks. We will determine if AAV vector integration occurs at breakage sites by rescuing retrovirus shuttle vectors containing I-Scel recognition sites through cloning in bacteria, and evaluate the sequence at the breakage site. Finally, we plan to disrupt genes involved in homologous recombination and DSBR (Rad52, Rad54, and Ku70) using homologous integration of AAV vectors and define cellular pathways involved in homologous and non-homologous AAV vector integration. More information about the mechanism and effect of AAV vector transduction will reveal safety issues for ongoing clinical trials, as well as allow improvement of these vectors for continued use as gene delivery vehicles.